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Effects of imatinib mesylate in osteoblastogenesis

2009; Elsevier BV; Volume: 37; Issue: 4 Linguagem: Inglês

10.1016/j.exphem.2008.12.008

1873-2399

Daniele Tibullo, Cesarina Giallongo, Piera La Cava, Salvatore Berretta, Fabio Stagno, Annalisa Chiarenza, Concetta Conticello, Giuseppe A. Palumbo, Francesco Di Raimondo,

Myeloproliferative Neoplasms: Diagnosis and Treatment

Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 μM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-κ B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p < 0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p < 0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis. Imatinib mesylate (IM), a tyrosine kinase inhibitor currently used in chronic myeloid leukemia (CML), may also affect the growth of other cellular systems besides CML cells. Because it has been reported that IM may affect bone tissue remodeling, we evaluated the effects of IM on osteoblatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs). After 21 days of culture, hBM-MSCs treated with IM (1 μM) alone or osteogenic medium (OM) + IM showed changes in morphology with evidence of extracellular mineralization and increased mRNA expression of osteogenic markers, such as RUNX2, osteocalcin (OCN), and bone morphogenetic protein (BMP-2). We also observed that levels of OCN and the osteoprotegerin (OPG)/receptor activator of nuclear factor-κ B ligand (RANKL) ratio (OPG/RANKL ratio) were increased in the surnatant of the 21-day culture with IM or OM + IM compared to controls (p < 0.005). In addition, we found that in 46 serum samples collected from CML patients treated with IM for 3 to 24 months, the OPG/RANKL ratio increased after 3 and 6 months (p < 0.004) returning back to the basal level after 24 months of IM treatment. In these patients, OCN levels were low at diagnosis but they increased throughout the IM treatment, approaching normal levels at 24 months of IM therapy. In summary, our data show that IM increases mRNA expression of osteogenic markers in hBM-MSCs and increases the OPG/RANKL ratio and the OCN levels both in surnatant of hBM-MSCs cultured with IM and in serum of patients treated with IM, thus indicating that IM potentially favors osteoblastogenesis. Imatinib mesylate (IM; Glivec, Novartis, Basel, Switzerland) belongs to a new generation of rationalized developed drugs. It is a tyrosine kinase inhibitor presenting as original target the chimeric protein BCR-ABL, derived from the molecular juxtaposition of two genes, BCR and ABL, on the Philadelphia chromosome in chronic myeloid leukemia (CML) [1Berman E. Nicolaides M. Maki R.G. et al.Altered bone and mineral metabolism in patients receiving imatinib mesylate.N Engl J Med. 2006; 354: 2006-2013Crossref PubMed Scopus (242) Google Scholar, 2Capdeville R. Buchdunger E. Zimmermann J. Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug.Nat Rev Drug Discov. 2002; 1: 493-502Crossref PubMed Scopus (1219) Google Scholar]. IM is also used to treat gastrointestinal stromal tumors by inhibiting stem cell factor receptor (c-kit) tyrosine kinases [3Oswald J. Boxberger S. Jorgensen B. et al.Mesenchymal stem cells can be differentiated into endothelial cells in vitro.Stem Cells. 2004; 22: 377-384Crossref PubMed Scopus (1099) Google Scholar, 4Buchdunger E. Cioffi C.L. Law N. et al.Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors.J Pharmacol Exp Ther. 2000; 295: 139-145PubMed Google Scholar, 5Verweij J. van Oosterom A. Blay J.Y. et al.Imatinib mesylate (STI-571 Glivec, Gleevec) is an active agent for gastrointestinal stromal tumours, but does not yield responses in other soft-tissue sarcomas that are unselected for a molecular target. Results from an EORTC Soft Tissue and Bone Sarcoma Group phase II study.Eur J Cancer. 2003; 39: 2006-2011Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar]. Inhibition of other protein kinase receptor autophosphorylation was demonstrated for FMS-like tyrosine kinase 3, c-FMC, v-fms, platelet-derived growth factor receptor (PDGFR)–β, and v-SRC family kinases, although at a much higher dosage [2Capdeville R. Buchdunger E. Zimmermann J. Matter A. Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug.Nat Rev Drug Discov. 2002; 1: 493-502Crossref PubMed Scopus (1219) Google Scholar, 6Kurzrock R. Kantarjian H.M. Druker B.J. Talpaz M. Philadelphia chromosome-positive leukemias: from basic mechanisms to molecular therapeutics.Ann Intern Med. 2003; 138: 819-830Crossref PubMed Scopus (268) Google Scholar]. Patients affected by CML are treated for an indefinite time with IM. Therefore, it is possible that this drug, in a long run, may have some effects on systems other than the hematopoietic bone marrow. In particular, it has been demonstrated that long-term treatment of CML patients with IM is associated with altered bone and mineral metabolism with reduction of bone remodeling [1Berman E. Nicolaides M. Maki R.G. et al.Altered bone and mineral metabolism in patients receiving imatinib mesylate.N Engl J Med. 2006; 354: 2006-2013Crossref PubMed Scopus (242) Google Scholar]. In normal individuals, bone remodeling occurs throughout entire life to maintain skeletal integrity. This process is carried out by osteoblasts (OB) and osteoclasts (OC), which are responsible, respectively, for bone formation and resorption by strictly interacting with each other. OBs are continuously recruited from stem and progenitor cells present in bone marrow during bone-formation phase of skeletal remodeling. While the identity of these stem cells has not been unequivocally determined, it is generally accepted that they are represented by clonogenic multipotential cells in the bone marrow stroma known as human bone marrow mesenchymal stem cells (hBM-MSCs) [7Rickard D.J. Kassem M. Hefferan T.E. Sarkar G. Spelsberg T.C. Riggs B.L. Isolation and characterization of osteoblast precursor cells from human bone marrow.J Bone Miner Res. 1996; 11: 312-324Crossref PubMed Scopus (332) Google Scholar, 8Bianco P. Riminucci M. Gronthos S. Robey P.G. Bone marrow stromal stem cells: nature, biology, and potential applications.Stem Cells. 2001; 19: 180-192Crossref PubMed Scopus (1710) Google Scholar]. On the contrary, osteoclasts are large multinucleated cells located on endosteal bone surfaces derived from monocytic progenitor cells [9Bahar H. Benayahu D. Yaffe A. Binderman I. Molecular signaling in bone regeneration.Crit Rev Eukaryot Gene Expr. 2007; 17: 87-101Crossref PubMed Google Scholar, 10Fujikawa Y. Quinn J.M. Sabokbar A. McGee J.O. Athanasou N.A. The human osteoclast precursor circulates in the monocyte fraction.Endocrinology. 1996; 137: 4058-4060Crossref PubMed Scopus (297) Google Scholar]. In order to maintain a bone homeostasis, osteoclastic bone resorption and osteoblastic bone formation must be tightly regulated and imbalances between OB and OC activity result in development of skeletal abnormalities. Recently, it has been shown that osteoblasts regulate differentiation of osteoclasts by two factors, the receptor activator of nuclear factor-κB ligand (RANKL), and the osteoprotegerin (OPG). RANKL is a transmembrane ligand expressed by osteoblasts and bone marrow stromal cells [11Lacey D.L. Timms E. Tan H.L. et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4546) Google Scholar, 12Simonet W.S. Lacey D.L. Dunstan C.R. et al.Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.Cell. 1997; 89: 309-319Abstract Full Text Full Text PDF PubMed Scopus (4261) Google Scholar, 13Kong Y.Y. Yoshida H. Sarosi I. et al.OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.Nature. 1999; 397: 315-323Crossref PubMed Scopus (2799) Google Scholar]. Following binding to RANK, a receptor for osteoclast differentiation, activation, and survival, RANKL induces osteoclastogenesis. OPG, also produced by osteoblasts and marrow stromal cells, acts as a decoy receptor for RANKL, thus regulating bone metabolism [7Rickard D.J. Kassem M. Hefferan T.E. Sarkar G. Spelsberg T.C. Riggs B.L. Isolation and characterization of osteoblast precursor cells from human bone marrow.J Bone Miner Res. 1996; 11: 312-324Crossref PubMed Scopus (332) Google Scholar]. The mechanism of bone remodeling inhibition by IM is not completely understood, but it has already been demonstrated that IM inhibits the differentiation and function of osteoclasts at concentrations within the therapeutic dose range [14Dewar A.L. Farrugia A.N. Condina M.R. et al.Imatinib as a potential antiresorptive therapy for bone disease.Blood. 2006; 107: 4334-4337Crossref PubMed Scopus (66) Google Scholar, 15El Hajj Dib I. Gallet M. Mentaverri R. Sevenet N. Brazier M. Kamel S. Imatinib mesylate (Gleevec) enhances mature osteoclast apoptosis and suppresses osteoclast bone resorbing activity.Eur J Pharmacol. 2006; 551: 27-33Crossref PubMed Scopus (58) Google Scholar]. On the other side, discordant data are present on the effect of IM on OBs. One study [16Fierro F. Illmer T. Jing D. et al.Inhibition of platelet-derived growth factor receptor beta by imatinib mesylate suppresses proliferation and alters differentiation of human mesenchymal stem cells in vitro.Cell Prolif. 2007; 40: 355-366Crossref PubMed Scopus (76) Google Scholar] indicates that IM is able to suppress proliferation of mesenchymal cells and favors their differentiation toward adipocytes at the expenses of osteoblastic formation. On the contrary, another study carried out on primary rat osteoblastic cells and a human osteoblastic cell line indicates that IM is able to stimulate the differentiation of OBs while inhibiting their proliferation and survival. Two recent clinical studies [17Fitter S. Dewar A.L. Kostakis P. et al.Long-term imatinib therapy promotes bone formation in CML patients.Blood. 2008; 111: 2538-2547Crossref PubMed Scopus (133) Google Scholar, 18Jonsson S. Olsson B. Ohlsson C. Lorentzon M. Mellstrom D. Wadenvik H. Increased cortical bone mineralization in imatinib treated patients with chronic myelogenous leukemia.Haematologica. 2008; 93: 1101-1103Crossref PubMed Scopus (45) Google Scholar], by using an histomorphometric evaluation and a bone mineral density one, have confirmed that long-term IM therapy promotes bone formation and, although IM suppresses mesenchymal cell proliferation, it is able to promote OBs differentiation by inhibition of the PDGFRβ [17Fitter S. Dewar A.L. Kostakis P. et al.Long-term imatinib therapy promotes bone formation in CML patients.Blood. 2008; 111: 2538-2547Crossref PubMed Scopus (133) Google Scholar]. We, therefore, undertook a study in order to evaluate the in vitro effect of IM on OB generation and the in vivo modification of markers of bone formation in CML patients treated with IM. Mesenchymal stem cells (hBM-MSCs) were obtained from bone marrow samples of normal healthy adult bone marrow donors after informed consent. hBM-MSCs were isolated by density gradient (mononuclear fraction) cultured in low-glucose Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal calf serum (Biochrom, Cambridge, UK). After 24 hours, no-adherent cells were eliminated and adherent cells were replaced with fresh medium. The immunophenotype (characteristically CD73+, CD90+, CD105+, CD34−, CD45−) and the ability to form fibroblastoid colony-forming units (CFU-F) were checked in all samples. Osteogenic differentiation of hBM-MSCS was induced by incubating 70% to 80% confluent cultures in standard condition (SC) (Dulbecco's modified Eagle's medium +10% fetal calf serum +100 U/mL penicillin +100 μg/mL streptomycin). Briefly, after primary culture in SC and expansion for two passages, the cells were trypsinized and replated onto 25 cm2 tissue culture flask at a density of 105 cells; therefore, cells were incubated with SC or IM dissolved in phosphate-buffered saline or osteogenic medium (OM) (0.2 mM ascorbic acid [Sigma, St Louis, MO, USA], 0.1 μm dexamethasone [Sigma], and 10 mM β-glycerophosphate] or the combination of both. Medium (with SC, IM, OM, or IM + OM) was replaced every 3 to 4 days. The concentration of IM used in experimental conditions was 1 μM, which is the dose easily reachable in the plasma of patients treated with IM [19Peng B. Hayes M. Resta D. et al.Pharmacokinetics and pharmacodynamics of imatinib in a phase I trial with chronic myeloid leukemia patients.J Clin Oncol. 2004; 22: 935-942Crossref PubMed Scopus (412) Google Scholar]. Mineralization was determined using Alizarin Red S (Sigma) staining and phase-contrast microscopy at 21days of culture. Cells were incubated with 2% alizarin red with pH 4.2 for 10 minutes, subsequently washed with distilled water and then observed with phase-contrast microscopy to examine cell morphology and to verify the presence of mineralized nodules. Expression of osteoblast-associated genes, such as osteocalcin (OCN), runt-related transcription factor 2 (Runx2)/Cbfa1, and bone morphogenetic protein (BMP-2), were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Cultured cell layers in different conditions (SC, IM, OM, or IM + OM) were rinsed at 21 days with cold phosphate-buffered saline and immediately lysed using Trizol Reagent (Qiagen, Hilden, Germany). Total RNA was isolated and treated by RNase-free DNase I and quantified by ultraviolet spectrophotometry. For RT-PCR analysis of mRNA expression, 1.0 μg total RNA (in 20 μL reaction volume) was reverse-transcribed using reverse transcriptase (Roche Diagnostics, Mannheim, Germany) and oligo-dT primers in a standard reaction. The resultant cDNA was then used as template for PCR amplification (in 25 μL reaction volume) of glyceraldehyde-3-phosphate dehydrogenase, OCN, Runx2/Cbfa1, and BMP-2. The primers used in this investigation are listed in Table 1 and all primer sequences were determined through established GenBank sequences. Amplification of glyceraldehyde-3-phosphate dehydrogenase was used as a control for assessing PCR efficiency. Each reaction was evaluated by 2% agarose gel electrophoresis. Ethidium bromide–stained gels were digitally photographed. Semi-quantitative evaluation by densitometry analysis was performed by Scion Image software (Scion Corporation, Frederick, MD, USA).Table 1Nucleotide sequences of primers used for quantitative reverse transcription polymerase chain reaction detectionGenePrimer sequencesForwardReverseBMP25′-AGACCTGTATCGCAGGCACT-3′5′-CCAACCTGGTGTCCAAAAGT-3′OCN5′-GGCAGCGAGGTAGTGAAGAG-3′5′-AGCAGAGCGACACCCTAGAC-3′Runx25′-GGTTCCAGCAGGTAGCTGAG-3′5′-GCCTACAAAGGTGGGTTTGA-3′BMP = bone morphogenetic protein; OCN = osteocalcin; Runx2 = runt-related transcription factor 2. Open table in a new tab BMP = bone morphogenetic protein; OCN = osteocalcin; Runx2 = runt-related transcription factor 2. By using a specific enzyme-linked immunosorbent assay (ELISA) test, according to manufacturer's recommendations, we evaluated the OCN levels (BioSource International, Inc., Camarillo, CA, USA), the OPG (osteoprotegerin) and RANKL levels in the surnatant culture (BioVendor Laboratory Medicine, Modrice, Czech Republic). OPG and RANKL levels were also assayed in serum of patients affected by CML and treated with IM. Healthy volunteers were used as control. The OPG/RANKL ratio was calculated as it represents an indicator of bone turnover and metabolism and an increase of this ratio indicates a shift toward osteogenesis [20Lee L. Liu J. Manuel J. Gorczynski R.M. A role for the immunomodulatory molecules CD200 and CD200R in regulating bone formation.Immunol Lett. 2006; 105: 150-158Crossref PubMed Scopus (19) Google Scholar]. Results are expressed as mean ± standard error of mean and the statistical analysis was performed using analysis of variance. A value of P < 0.05 was considered as significant. hBM-MSCs were cultured with either OM medium alone, or IM 1 μM alone or a combination of both for 21 days and compared to untreated cultures. In comparison to control cultures, hBM-MSCs placed in OM, IM, and IM + OM exhibited changes in cell morphology already detectable after 7 days of culture. The cell morphology changed from a spindle-shaped fibroblastic appearance to a rounder, more cuboidal shape and the cells formed an extensive network of dense multilayered nodules (extracellular mineralization). Extracellular mineralization capacity was confirmed by Alizarin Red S staining of the induced cells after 21 days of culture (Fig. 1). This phenomenon was present in hBM-MSC cultures with OM or IM alone but it was more evident with the combination. To confirm the induction of osteogenesis, cultured cells were also examined by RT-PCR for the expression of the osteoblast-related genes OCN, Runx2/cfba1, and BMP-2 (Fig. 2). At 21 days, the levels of OCN, Runx2/cfba1, and BMP-2 expression were increased in cells treated with OM, IM, and OM + IM in respect to control (Table 2). In particular, levels of BMP-2 were significantly higher in IM-treated cultures in respect to control (p < 0.0007) and they were also higher in OM + IM-treated culture than in OM alone (p < 0.002). The same differences are applied to OCN and Runx2/Cfba1, although at less significant p values (Fig. 2).Table 2Value of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) protein levels and OPG/RANKL ratio in sera of healthy donors and chronic myeloid leukemia patients at diagnosis and throughout treatment with imatinib mesylateHealthy donorsDiagnosis3 months6 months24 monthsOPG (pmol/mL)2.57 ± 0.3418.53 ± 4.824.09 ± 3.0923.02 ± 3.88.51 ± 3.33RANKL (pmol/mL)2.53 ± 0.372.96 ± 1.61.33 ± 0.81.91 ± 0.92.8 ± 3.29OPG/RANKL ratio1.11 ± 0.0836.253 ± 1.5118.40 ± 5.812.05 ± 3.33.070 ± 0.70 Open table in a new tab OCN levels and OPG/RANKL ratio were analyzed in culture surnatant by ELISA after 21 days of culture (Fig 3). Level of OCN were 1.417 ± 0.03177 ng/mL in control cultures, 1.756 ± 0.0502 ng/mL in OM cultures, 2.267 ± 0.1096 ng/mL in presence of IM 1 μM (p < 0.0001 vs control), and 4.028 ± 0.08159 ng/mL with the combination of OM + IM (p < 0.0001 vs OM alone). Further on, OPG/RANKL ratio was increased in hBM-MSCs culture after 21 days of treatment with IM in respect to control (75.76 ± 1.668 vs 51.96 ± 2.126, P<0.0001) and it was also significantly higher in OM + IM compared to cultures with OM alone (73.14 ± 4.101 vs 62.10 ± 1.885; p < 0.02). We also evaluated by ELISA, the serum OPG/RANKL ratio and the serum levels of OCN in IM-treated CML patients. A total of 46 serum samples from CML patients were collected, while 10 healthy volunteers, comparable for age and gender, were used as control. Samples were representative of 12 patients at diagnosis, 9 patients each after 3 and 6 months of treatment with IM 400 mg, and 16 patients after 24 month of treatment. In eight of these patients, the OPG/RANKL ratio and the OCN levels were evaluated at diagnosis and throughout the treatment. All the treated patients were in complete hematologic remission at each time point postdiagnosis, and 12 of them were also in major molecular remission at 24 months of treatment. We found that the OPG/RANKL ratio in patients at diagnosis was higher in comparison to healthy voluntaries (6.253 ± 1.519 vs 1.110 ± 0.08340; p > 0.05) and the ratio further increased after 3 (18.40 ± 5.891; p < 0.02) and 6 months (12.05 ± 3.3), but it returned at the basal level after 24 months of treatment with IM (p < 0.0001) (Fig. 4). The increase of the OPG/RANKL ratio was sustained mainly by an increase in the OPG levels and to a minor extent by a decrease in RANKL levels (Table 2). OCN levels in patients at diagnosis were low in comparison to healthy donors (1.392 ± 0.0922 ng/mL vs 11.37 ± 0.3836 ng/mL, p < 0.02), but they showed a step by step increase throughout the IM treatment (4.166 ± 1.150 ng/mL, 5.162 ± 1.827 ng/mL, 9.240 ± 0.7962 ng/mL after 3, 6, and 24 months of therapy, respectively (p < 0.0001), so that at 24 months OCN levels in IM-treated CML patients were close to normal ones. In this report, we have demonstrated that IM induces hBM-MSCs to differentiate toward the osteoblastic lineage. Compared to control, in cultures treated with IM 1 μM for 21 days, we have observed an increase in extracellular mineralization and an increase in osteogenic markers (OCN, Runx2/cfba1, and BMP-2) mRNA. OCN levels and OPG/RANKL ratio were also increased in surnatant of cultures treated with IM. In addition, we have shown that CML patients treated with conventional doses of IM have a transient elevation of OPG levels which, together with a slight reduction of RANKL levels, was responsible of a significant increase of the OPG/RANKL ratio that returns to the basal level after 24 months. It is possible that this late reduction of OPG/RANKL ratio depends on the coordination of osteoclast with osteoblasts activities that are reciprocally and tightly regulated in order to maintain bone homeostasis. Incidentally, we have found that an increase of the OPG/RANKL ratio is already present in patients affected by CML at diagnosis compared to healthy controls. This finding deserves further studies and leads to the hypothesis that CML patients may be more susceptible to alteration of bone metabolism than normal subjects. However, in our series, a contribution of the disease itself to the increase in the OPG/RANKL ratio, in the time points subsequent to diagnosis, can be excluded because all samples came from patients who were at least in complete hematological response. We also observed that OCN levels at diagnosis were lower than healthy controls, but were progressively increased in CML patients treated with imatinib. There are two contrasting reports in literature on the osteocalcin serum levels in CML patients treated with imatinib. Berman et al. [1Berman E. Nicolaides M. Maki R.G. et al.Altered bone and mineral metabolism in patients receiving imatinib mesylate.N Engl J Med. 2006; 354: 2006-2013Crossref PubMed Scopus (242) Google Scholar] found a reduced level compared to healthy controls, while Grey et al. [21Grey A. O'Sullivan S, Reid IR, Browett P. Imatinib mesylate, increased bone formation, and secondary hyperparathyroidism.N Engl J Med. 2006; 355: 2494-2495Crossref PubMed Scopus (51) Google Scholar] observed that OCN level increased during IM treatment compared to baseline. We confirmed the observation of Grey et al., but our findings are also consistent with Berman et al.'s report because we have observed in our patients that OCN serum levels were low at diagnosis and showed a slow increase during IM treatment, approaching normal values after 24 months only, thus explaining the finding of Berman et al. [1Berman E. Nicolaides M. Maki R.G. et al.Altered bone and mineral metabolism in patients receiving imatinib mesylate.N Engl J Med. 2006; 354: 2006-2013Crossref PubMed Scopus (242) Google Scholar] of a low OCN levels when they are measured too early. At this time, we do not have a clear explanation for the low OCN levels at diagnosis, but an induction of OCN-mRNA has already been reported in imatinib-treated cell lines [22Wihlidal P. Karlic H. Pfeilstocker M. Klaushofer K. Varga F. Imatinib mesylate (IM)-induced growth inhibition is associated with production of spliced osteocalcin-mRNA in cell lines.Leuk Res. 2008; 32: 437-443Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. Recent clinical studies have already indicated that, within 3 months of starting imatinib, CML patients have several alterations in mineral metabolism [1Berman E. Nicolaides M. Maki R.G. et al.Altered bone and mineral metabolism in patients receiving imatinib mesylate.N Engl J Med. 2006; 354: 2006-2013Crossref PubMed Scopus (242) Google Scholar], increase of specific markers of bone formation [21Grey A. O'Sullivan S, Reid IR, Browett P. Imatinib mesylate, increased bone formation, and secondary hyperparathyroidism.N Engl J Med. 2006; 355: 2494-2495Crossref PubMed Scopus (51) Google Scholar], and, in the long run, significant increase of trabecular bone volume [17Fitter S. Dewar A.L. Kostakis P. et al.Long-term imatinib therapy promotes bone formation in CML patients.Blood. 2008; 111: 2538-2547Crossref PubMed Scopus (133) Google Scholar], and increased cortical bone mineralization [18Jonsson S. Olsson B. Ohlsson C. Lorentzon M. Mellstrom D. Wadenvik H. Increased cortical bone mineralization in imatinib treated patients with chronic myelogenous leukemia.Haematologica. 2008; 93: 1101-1103Crossref PubMed Scopus (45) Google Scholar]. These findings do not demonstrate a direct cause-and-effect action of imatinib, because it could be argued that these effects could be just related to the progressive reduction of leukemic cells, which in some way might have altered the bone metabolism. However, in vitro studies, including our data, strongly suggest that the alteration of bone metabolism is indeed induced by imatinib. A study conducted on murine osteoblastic and preosteoblastic cells and on human cell lines, demonstrated that IM inhibits the proliferation and survival of preosteoblastic cells by its ability to inhibit PDGFR signaling, but through the same mechanism, authors have demonstrated that IM is able to promote osteoblastic differentiation, leading to the hypothesis that PDGF promotes OB expansion by blocking their differentiation while IM induces the opposite effect by interfering with PDGFR signaling [23O'Sullivan S. Naot D. Callon K. et al.Imatinib promotes osteoblast differentiation by inhibiting PDGFR signaling and inhibits osteoclastogenesis by both direct and stromal cell-dependent mechanisms.J Bone Miner Res. 2007; 22: 1679-1689Crossref PubMed Scopus (98) Google Scholar]. Another recent study [17Fitter S. Dewar A.L. Kostakis P. et al.Long-term imatinib therapy promotes bone formation in CML patients.Blood. 2008; 111: 2538-2547Crossref PubMed Scopus (133) Google Scholar] has shown that, while restraining mesenchymal cells proliferation, IM favors their differentiation toward the osteoblastic lineage. A central role of PDGF in the mechanism of osteoblastogenesis was suggested by the fact that authors observed a reduced mineral formation when PDGF was added to the cultures and this effect was counteracted by IM 3 μM. In addition, bone explanted cell cultured under osteogenic conditions in the presence of PDGFR inhibitors produced up to fourfold more mineralized matrix than untreated controls. However, it is not clear whether the effect of IM on osteoblastogenesis may be entirely due to inhibition of PDGFR signaling. We have shown that IM increased the mRNA expression of two important osteoblastogenic genes, such as BMP-2 and Runx2/cfba1. Indeed, recent data indicate that BMP-2 requires Runx2 for induction of osteoblastic differentiation [24Javed A. Bae J.S. Afzal F. et al.Structural coupling of Smad and Runx2 for execution of the BMP2 osteogenic signal.J Biol Chem. 2008; 283: 8412-8422Crossref PubMed Scopus (179) Google Scholar] and, on this basis, we are incline to hypothesize that other mechanisms besides the inhibition of the PDGFR, in particular, the BMP/Runx2 axis, may have a role in the imatinib-mediated induction of osteoblasts (Fig. 5). Runx2/cfba1 is a transcription factor required for in vivo bone formation [25Hassan M.Q. Tare R.S. Lee S.H. et al.BMP2 commitment to the osteogenic lineage involves activation of Runx2 by DLX3 and a homeodomain transcriptional network.J Biol Chem. 2006; 281: 40515-40526Crossref PubMed Scopus (177) Google Scholar] and its overexpression has been reported to stimulate osteoblast differentiation in a variety of cell types, including fibroblasts, hBM-MSCs, and myoblasts [26Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3578) Google Scholar, 27Xiao Z.S. Hinson T.K. Quarles L.D. Cbfa1 isoform overexpression upregulates osteocalcin gene expression in non-osteoblastic and pre-osteoblastic cells.J Cell Biochem. 1999; 74: 596-605Crossref PubMed Scopus (97) Google Scholar]. Although Runx2/cfba1 expression is regulated through several pathways including Wnt/β-catenin [28Gaur T. Lengner C.J. Hovhannisyan H. et al.Canonical WNT signaling promotes osteogenesis by directly stimulating Runx2 gene expression.J Biol Chem. 2005; 280: 33132-33140Crossref PubMed Scopus (881) Google Scholar] and transforming growth factor–β/Smad3 signaling [29Lee K.S. Kim H.J. Li Q.L. et al.Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12.Mol Cell Biol. 2000; 20: 8783-8792Crossref PubMed Scopus (748) Google Scholar], in our hypothesis the pathways of interest are mainly represented by PDGF-R and BMP receptors. BMP ligands, such as BMP-2 (in our experiments increased by IM), bind BMP receptors, which, once activated by phosphorylation, regulate target gene expression [30Herpin A. Cunningham C. Cross-talk between the bone morphogenetic protein pathway and other major signaling pathways results in tightly regulated cell-specific outcomes.FEBS J. 2007; 274: 2977-2985Crossref PubMed Scopus (84) Google Scholar], including Runx2/cfba1 [31Phimphilai M. Zhao Z. Boules H. Roca H. Franceschi R.T. BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype.J Bone Miner Res. 2006; 21: 637-646Crossref PubMed Scopus (284) Google Scholar]. Experimental evidences indicate that BMP signaling is physiologically balanced by mitogen-activated protein kinase (MAPK) signals and that the two pathways converge on Smad1. Phosphorylation of Smad1 by MAPKs, such as Erk, Jnk, and p38 inhibits BMP signaling [32Kretzschmar M. Doody J. Massague J. Opposing BMP and EGF signalling pathways converge on the TGF-beta family mediator Smad1.Nature. 1997; 389: 618-622Crossref PubMed Scopus (763) Google Scholar, 33Aubin J. Davy A. Soriano P. In vivo convergence of BMP and MAPK signaling pathways: impact of differential Smad1 phosphorylation on development and homeostasis.Genes Dev. 2004; 18: 1482-1494Crossref PubMed Scopus (129) Google Scholar]. Then, inhibition of PDGFR by IM decreases activity of MAPKs, which in turn cannot inhibit Smad1. As a consequence, BMP-2 can activate the BMP/Runx2 axis and allow the expression of BMP target genes (Fig. 5). In conclusion, we have confirmed that in IM-treated CML patients, there is a transient increase in bone-formation markers [21Grey A. O'Sullivan S, Reid IR, Browett P. Imatinib mesylate, increased bone formation, and secondary hyperparathyroidism.N Engl J Med. 2006; 355: 2494-2495Crossref PubMed Scopus (51) Google Scholar] that justifies the recent clinical observation of an increase in trabecular bone volume [17Fitter S. Dewar A.L. Kostakis P. et al.Long-term imatinib therapy promotes bone formation in CML patients.Blood. 2008; 111: 2538-2547Crossref PubMed Scopus (133) Google Scholar] and cortical bone mineralization [18Jonsson S. Olsson B. Ohlsson C. Lorentzon M. Mellstrom D. Wadenvik H. Increased cortical bone mineralization in imatinib treated patients with chronic myelogenous leukemia.Haematologica. 2008; 93: 1101-1103Crossref PubMed Scopus (45) Google Scholar] in these patients. Although these effects could be related just to achievement of remission and, therefore, to the elimination of leukemic cells that could interfere with bone metabolism, our in vitro observation that IM increases the capacity of hBM-MSCs to differentiate into osteogenic lineage; induces expression of osteoblast-related genes; and increases levels of OCN and OPG/RANKL ratio in culture surnatant, strongly supports the hypothesis that at least some parts of the bone modification are really induced by imatinib. This imatinib "side effect" actually could turn to be a beneficial one, because, although it is difficult to foresee the long-term clinical consequences, it is possible that CML patients treated for a long time with IM could have a reduction of the physiological osteoporosis that occurs in elderly patients. This study has been supported in part by A.I.L. (Associazione Italiana contro le Leucemie) sezione di Catania and by FON.CA.NE.SA. (Fondazione Catanese per lo Studio delle Malattie Neoplastiche del Sangue).